Method for the chromatographic fractionation of plasma or serum, preparations, so obtained, and their use

ABSTRACT

The invention relates to the fractionation of plasma or serum into at least one albumin fraction and one immunoglobulin fraction by hydrophobic interaction chromatography. The fractionation is carried out using an incremental salt gradient, especially an ammonium sulfate buffer. The invention also relates to preparations obtained by using said method and to their use.

This application is a 371 of PCT/EP00/05827 filed on Jun. 23, 2000.

The invention relates to a method, described in the claims, for thechromatographic fractionation of plasma or serum, especially of humanplasma and/or serum, at a hydrophobic interaction phase by means of astepwise salt gradient, as well as to the protein preparations, preparedtherefrom, and their use.

The first attempts to fractionate human plasma industrially were carriedout already in the 1940s. The methods used are based on theprecipitation of protein fractions by means of alcohol, different plasmaproteins being obtained in an enriched form by varying the pH, the ionicstrength, the temperature and the alcohol concentrations in thesefractions.

The fractions, which are obtained by this process and containimmunoglobulins and albumin, are of particular importance for thetherapeutic application. An overview of the above-mentioned methods isgiven in the review articles (J. E. More and M. J. Harvey, BloodSeparation and Plasma Fractionation, Wiley-Liss, New York, 1991,261–306, and P. Kistler and H. Friedl, Methods of Plasma ProteinFractionation, Academic Press, London, 1980, pages 3 to 15).

Aside from alcohol, other precipitating reagents, such as ammoniumsulfate and polyethylene glycol, are used to recover plasma fractions(A. P. Phillips, K. L. Martin and W. H. Horton, The choice of methodsfor immunoglobulin IgG purification. Yield and Purity of AntibodyActivity. J. of Immunological Methods, 74 (1984) 385–393. A. Polson etal., The Fractionation of Protein Mixtures by Linear Polymers of HighMolecular Weight. Biochem. Biophys. Acta, 82 (1964) 463–475; P. D.Gorevich et al., Methods of Enzymology, vol. 116, 1985, 3–25; DE2936047C2).

In principle, all precipitation methods for isolating plasma proteinshave some disadvantages. The reagents, used for the precipitation, bringabout a partial denaturation of the separated proteins. This can be seenby the formation of aggregates and the incompatibilities associatedtherewith during the therapeutic application. Further expensive cleaningsteps are required in order to obtain compatible preparations from theprotein fractions so obtained.

A further disadvantage of the methods consists therein thatprecipitation reactions, especially in the case of mixtures as complexas human plasma, are never quantitative. This and the additionalcleaning steps required lead to appreciable losses in yield.

These methods therefore do not represent an optimum utilization of thevaluable starting material, particularly of human plasma or human serum.

For this reason, attempts were made to carry out the plasmafractionation with gentler methods and higher yields. For this purpose,adsorptive methods are available.

Special properties of proteins, such as charge, hydrophobicity, size andcharacteristic binding properties are utilized to bind the proteins tosuitable ligands. It is particularly advantageous if these ligands arebound to a water-insoluble, stationary carrier. The binding of theproteins to the substituted carrier can be carried out in batch methodsor as a column chromatographic method, the latter having proved to beparticularly advantageous. Anionic and cationic exchangers, affinity andhydrophobic phases, as well as exclusion chromatographic materials wereused for the chromatographic isolation of plasma proteins.

These chromatographic methods were previously used primarily for thefine purification of precipitated plasma fractions.

Some examples are listed in the following.

In the EP 0447585B1, the isolation of immunoglobulin G from Cohn PasteII is described.

The EP 0352500B1 discloses the production of immunoglobulin M from PasteIII, which has been precipitated with alcohol.

Transferrin (DE 3733181C1, α₁-antitrypsin (EP 0717049A1, U.S. Pat. No.5,610,285) and antithrombin III (EP 0844254A2) can be isolated from CohnPaste IV with the help of different chromatographic methods.

Cohn Paste V is the starting material for the, the chromatographicpreparation of albumin (EP 0792887A1) as well as for purifying α₁ acidicglycoprotein (U.S. Pat. No. 5,739,293).

Likewise, alcohol containing supernatant solutions of the Cohnfractionation can be used as starting material for a chromatographicpreparation of albumin (EP 04022058B1).

A combination of alcohol precipitation and chromatography for obtainingplasma proteins is disclosed in U.S. Pat. No. 5,138,034.

The direct recovery of immunoglobulin G and albumin from human plasma,by means of ion exchange chromatography without a prior precipitationand separation of a precipitate is described in the DE 3640513C2 and theWO 94/29334.

A survey of the use of chromatographic methods for recovering proteinsfrom human plasma may be found in J. M. Curling, Separation of PlasmaProteins, Pharmacia Fine Chemicals AB, Uppsala, Sweden, 1993.

As already mentioned above, all the methods described have significantdisadvantages. For example, all plasma proteins, obtained byprecipitation reactions, lead to considerable losses in yield andrequire additional, expensive purification steps, in order to make thetherapeutic application possible. In addition, a great technical effortis required for cooling the reaction vessels, centrifuges or filters.

The products, obtained by means of ion exchange chromatography fromplasma, require expensive pre-working up of the human plasma.

For carrying out the ion exchange chromatography, the plasma must beadjusted to do a defined, low ionic strength. This can be achieved bydiluting or re-buffering.

Large volumes result here, which limit the amount of plasma, which canbe processed on an industrial scale. Moreover, the fibrinogen, containedin the human plasma, must be removed by an additional step before thechromatography, in order to prevent blockage of the column.

S. Goheen et al., in the J. of Chromatography, 326 (1985), 235–241,describe a method for fractionating human serum. For this purpose, aBio-Gel TSK Phenyl-5PW column is charged with the starting solution andsubsequently eluted at 0° C. by means of a linear ammonium sulfategradient in 0.1M sodium phosphate buffer. Such a linear elution can becarried out only on an analytical scale, since otherwise, when chargingthe starting solution and using said initial concentration of 1.7M, thecolumn would become blocked if one were to work on a preparative scale.Moreover, it is necessary to work at 0° C., in order to improve theresolution of the chromatogram. Because of the technical effortinvolved, such a procedure cannot be transferred to a process on apreparative scale.

It is an object of the invention to provide to a method, which can becarried out economically on an industrial scale, for the fractionationof plasma, especially of human plasma, or of serum, especially of humanserum, which furnishes native, unmodified plasma proteins in high yield.For this method, precipitating and dissolving therapeutically relevantproteins shall be largely avoided and the process shall take place atroom temperature.

Pursuant to the invention, this objective is accomplished owing to thefact that plasma or serum, especially of human origin, preferably aplasma or serum, which has been freed from the clotting factor VIIIand/or the clotting factors of the PPSB complex, especially of humanorigin, is chromatographed at a hydrophobic phase using a stepwise saltgradient. Especially ammonium sulfate is suitable as salt.

Surprisingly, it has been found that, with the help of the hydrophobicinteraction chromatography, plasma or serum can be separated at leastinto an immunoglobulin-containing fraction and an albumin-containingfraction on a preparative scale and that the chromatography can becarried out at room temperature without the use of expensive coolingequipment.

The inventive method is described in greater detail in the following.

1. Fractionation

Pursuant to the invention, (human) plasma or (human) serum can be used,which may be freed from clotting factors. Moreover, animal plasma orserum with or especially without clotting factors can also be used. Thestarting material can be obtained from a normal donor pool as well asfrom selected donors with high antibody titers against viral, bacterialor cellular antigens. In the case of selected starting materials(hyperimmunoglobulin), preferably those with high titers against CMV,hepatitis B, chickenpox, tetanus or anti-D are selected.

As starting material for the inventive method, preferably a plasma,particularly a human plasma or human serum, which has been freed fromclotting factors, is used. The separation of the valuable clottingfactors from plasma, which themselves are of great therapeutic benefit,is known.

For example, the cryo-precipitate, which has been obtained from athawing process, is used as starting material for the preparation offactor VIII concentrates and the factors II, VII, IX and X (PPSBcomplex) are isolated by adsorption on an ion exchanger and, in purifiedform, are also used therapeutically.

Therefore, for obtaining the preferred starting material for theinventive method, a plasma, especially one obtained according to theguidelines of the blood donor system by means of plasmapheresis, can bethawed at +4° C. and the cryo-precipitate removed by centrifugation.Factor VIII can be produced from this. The clotting factors of PPSBcomplex are separated by adsorption on an ion exchanger, such as across-linked to dextran, substituted with diethylaminoethyl groups (suchas DEAE Sephadex® A50).

Preferably, sufficient gradient salt per liter, solid ammonium sulfatein the case of ammonium sulfate, is added to the starting material, withor without clotting factors, so that, after several hours of stirring atroom temperature, such as 3 to 20 hours, and, optionally, after theaddition of water for injection purposes, the salt solution, especiallythe ammonium sulfate solution, has a conductivity, which corresponds tothe starting concentration of salt, especially of ammonium sulfate,selected for the commencement of the chromatography. After the additionof 0.5 to 5% filter aid, such as standard Super Cell or a differentfilter aid such as perlite, harbolite or celite, the solution isfiltered. The filtrate is used as the starting solution for thechromatography. The hydrophobic interaction phase preferably isequilibrated to the desired initial concentration of salt, especially ofammonium sulfate corresponding to the concentration selected in thestarting solution.

The chromatographic separation is based on the interaction ofhydrophobic domains of the proteins molecules with hydrophobic groups onthe stationary chromatographic phase. Under physiological conditions,the hydrophobic groups of the proteins molecules are not freelyaccessible, so that binding to a hydrophobic interaction phase does nottake place. By adding the salt, especially ammonium sulfate, the hydratesheath of the proteins is decreased, so that the hydrophobic domains areavailable for a hydrophobic interaction.

The hydrophobicity of the stationary phase is selected pursuant to theinvention so that an interaction of certain proteins with the phase cantake place. The binding to the phase depends, on the one hand, on thehydrophobicity of the chromatographic matrix and, on the other, on thesalt concentration and especially the ammonium sulfate concentration ofthe solution. Since precipitation of the proteins takes place at a highsalt concentration and especially at a high ammonium sulfateconcentrations, the salt concentration, such as the ammonium sulfateconcentration, is selected according to the hydrophobicity of thechromatographic phase, so that the desired interactions take place at asalt concentration or an ammonium sulfate concentration, whichpredominantly does not lead to precipitation.

This is shown, for example, in Table 1, in which the proportion ofproteins, still in solution, is given as a function of the concentrationof ammonium sulfate. Satisfactory values are not obtained at values of1.4 moles/L of ammonium sulfate and above.

Accordingly, the concentration of salt, especially of ammonium sulfate,should be less than 1.4 moles/L.

Accordingly, for the effective separation of immunoglobulin and albuminfractions, the initial concentration of ammonium sulfate is less than1.4 moles/L, to which, if desired, the chromatography phase and thestarting solution are adjusted, washing is carried out preferably with abuffer of this salt concentration and the ammonium sulfate concentrationsubsequently is lowered.

Preferably, the separation is carried out if the high concentration is0.6 to less than 1.4 moles/L, particularly if it is 1.3 moles/L, moreparticularly if it is 0.6 to 1.2 moles/L, especially if it is 0.7 to 1mole/L and most particularly if it is 0.8 to 0.9 moles/L, and if thelowered concentration is 0.4 to 0 moles/L and especially 0.3 to 0moles/L.

With such a concentration gradient, initially an albumin-containingfraction and then an immunoglobulin-containing fraction is obtained. Thelatter may still contain lipids, which advantageously are removed. Forthis purpose, a further concentration step of the ammonium sulfate canbe used in the second step. Such a concentration step starts, forexample, at 0.4, especially 0.3 moles/L to 0.1 moles/L, particularly 0.3moles/L to 0.1 moles/L, more particularly 0.3 to 0.15 moles/L and mostparticularly 0.3 moles/L and the concentration during this stepsubsequently is lowered to below 0.1 moles/L to 0 moles/L. The lipidsare removed from the phase only at the lowest, optionally zero (0moles/L) concentration and with that, are removed from theimmunoglobulin fraction, which leaves the phase at higher ammoniumsulfate concentrations.

The chromatography is carried out under the usual conditions, known forsuch phases, such as a pH of, for example, 7.0 and sodium (hydrogen)phosphate buffer or trishydroxymethylaminomethane as eluant. Phosphatebuffers (such as 0.01 M NaH₂PO₄) are particularly preferred.

For the inventive process, the known materials are suitable ashydrophobic interaction phases. These include phenyl-substituted oralkyl-substituted phases based on copolymers of glycidyl methacrylateand ethylene glycol dimethacrylate, copolymers of polystyrene anddivinylbenzene or silica gel coated with dextran or silica gel.

Alkyl-substituted and, particularly, phenyl-substituted copolymers ofglycidyl methacrylate and ethylene glycol dimethacrylate are especiallysuitable.

These are commercially available under the trade names of TSK-Phenyl5PW® and Toyopearl-Phenyl 650®.

Pursuant to the invention, an initial ammonium sulfate concentration of,for example, 0.7 to 1.0 moles/L, preferably of 0.8 to 1.0 moles/L andparticularly of 0.9 moles/L is selected for the chromatographicseparation.

As is evident from Table 2, particularly good results are obtained atsuch concentrations for the chromatographic separation on TSK-PhenylSPW®.

As described above, the starting solution, adjusted to an ammoniumsulfate concentration of 0.9 moles/L, can be applied on the equilibratedhydrophobic chromatography phase. At this salt concentration, a fraction1 is obtained, which passes through the phase without being bound. Thebound proteins are eluted with 0.01 moles/L of sodium phosphate of pH7.0 from the phase as fraction 2, that is, the ammonium sulfateconcentration is reduced to here 0 moles/L.

The composition of the chromatographic fractions obtained is shown inTable 3. As can be seen, a fraction 1 is obtained, which is referred toas the albumin fraction and is very similar in composition to asupernatant II/III of the alcohol fractionation of Cohn. It contains theimportant proteins, namely albumin, transferrin, antithrombin III andα-antitrypsin. All immunoglobulins are contained almost quantitativelyin fraction 2, which therefore is similar to the composition of a pasteII/III obtained by the cold ethanol method.

As can furthermore seen from Table 3, lipids and lipoproteins are alsocontained in fraction 2. These can make it more difficult to processfraction 2 into therapeutically usable proteins solutions, so that ithas proven advantageous to integrate a further elution step in theprocess to obtain a third fraction. For this purpose, after fraction 1is obtained, one can start at the highest concentration, such as 0.9moles/L of ammonium sulfate and then continue working with a stepgradient. For example, fraction 2 can be isolated at 0.3 moles/L.

The protein composition of this immunoglobulin-containing Fraction 2does not differ format of the fraction described above (Table 3,Fraction 2), with the exception that the lipoprotein content is lessthan 5%.

The lipoprotein fraction remains bound to the chromatography phase at0.3 moles/L and higher ammonium sulfate concentrations and can be elutedfrom the phase with 0.01 moles/L of sodium phosphate at pH 7.0 asFraction 3. In other words, the ammonium sulfate concentration isreduced here to 0 moles/L.

Preferably, therefore, 3 fractions are separated during the inventive,hydrophobic interaction chromatography, namely initially the albuminfraction, then the immunoglobulin fraction and then the lipoproteinfraction.

The inventive process is shown diagrammatically in FIG. 1.

2. Recycling

Buffer solutions, containing buffer salt, such as ammonium sulfate, arerequired in appreciable amounts for the industrial scale application ofthe method. In order to minimize the consumption of this salt as much aspossible, a recycling method, which is shown in FIG. 2, is used pursuantto the invention.

For this method, the ammonium sulfate buffer 1, for example, which hasbeen adjusted to the desired (high) concentration, can be recycled aspermeate from the first fraction obtained, for example, by means ofsuitable ultrafiltration membranes, while the first fraction is beingcollected and concentrated continuously. For the purpose of separatingthe second and, optionally the third fraction, a buffer with therespectively desired concentration is then lowered, as stated pursuantto the invention, by mixing the ammonium sulfate buffer 1 with a salt,which is not a salt gradient, that is, with a salt other than theammonium sulfate buffer 2, or by using only the buffer 2, and the secondor third fraction is separated, depending, on the number of fractionsdesired.

Preferably, as described above, three fractions are produced by using astep gradient after separating Fraction 1, the ammonium sulfateconcentrations, given above, being selected.

The fractions can be concentrated continuously, for example, by means ofultrafiltration.

Preferably furthermore, the chromatographic phase is purified after eachcycle with sodium hydroxide solution from a reservoir 3, sterilizationtaking place at the same time.

For example, after the column is charged with the plasma proteinsolution, which has been adjusted to a buffer 1 and filtered, forobtaining Fraction 1, it is washed with buffer 1 (for example, 0.9moles/L of ammonium sulfate, 0.01 moles/L of NaH₂PO₄ of pH 7.0), inorder to obtain all of Fraction 1. The fraction, so obtained, iscollected and concentrated continuously using an ultrafiltration unitwith a cut-off of 10 KD. The permeate, obtained here, is returned to thereservoir for buffer 1.

To elute Fraction 2, the salt concentration or ammonium sulfateconcentration is reduced as indicated and, for example, a mixed buffercan be produced or a buffer 2, which does not contain any salt, such asammonium sulfate, is selected alone, depending on how many separationsteps are desired.

For a step gradient, for example, for eluting Fraction 2, buffer 1 (0.9moles/L of ammonium sulfate, 0.01 moles/L of NaH₂PO₄, pH 7.0) and buffer2 (0.01 moles/L of NaH₂PO₄, pH 7.0) can be mixed on-line in a ratio of,for example, 1:3. This mixing ratio results in an elution buffer forFraction 2 with the mixed concentration of, for example, 0.3 moles/L ofammonium sulfate, 0.01 moles/L of NaH₂PO₄, pH 7.0. The collectedFraction 2 is also concentrated on-line using an ultrafiltration unitwith a cut-off of 10–30 KD.

The lipoproteins are then eluted from the hydrophobic phase with buffer2 (0.01 moles/L of NaH₂PO₄, pH 7.0) and discarded.

This method can be varied by changing the buffer/salt concentrations andmixing ratios within the inventively deserved range and the usualconditions known to those skilled in the art for said chromatographyphases. For example, a 1.1 to 1.5 etc. mixture can be produced from abuffer 2 with a concentration of 1 mole/L.

When larger amounts are processed, several chromatographic cycles arecarried out. Preferably, the hydrophobic phase is therefore cleaned and,at the same time, sterilized with 1.0 moles/L of sodium hydroxide aftereach separation cycle. The sodium hydroxide solution is in reservoir 3.

3. Further Processing

Fractions 1 and 2, obtained from the hydrophobic phase using theinventive method, can be processed further into therapeutically usable,plasma protein solutions of high purity by known methods, such as thosedescribed in the publications mentioned above. Such preparations areused for respective deficiency or disease states. For example, in thecase of infections preparations, which were prepared pursuant to theinvention from selected starting material, such as those containing,anti-CMV or anti-HBs, anti-chickenpox, tetanus or anti-D, suchpreparations can be administered for the appropriate infection.

Amounts and forms of administration are known here. For example, thepreparations may be administered as injections or i.m. injection or i.v.infusion. In addition to the active ingredient, the pharmaceuticalcomposition may also contain the known inactive ingredients, whichinclude, for example, electrolytes, amino acids or sugars.

a) Immunoglobulin G

For example, especially an immunoglobulin G, one which is i.v.compatible, can be produced from the immunoglobulin fraction (Fraction2). For this purpose, known methods, such as those already described inDE 3640513C2 as well as EP-B 447 585, can be employed. This procedure isshown in FIG. 3 and comprises, essentially, anion exchangechromatography, optionally virus filtration, treatment with octanoicacid, cation exchange chromatography and the usual concentrating,filtering and sterilizing steps.

For example, by means of diafiltration, the concentrated Fraction 2 fromthe hydrophobic interaction chromatography can be re-buffered to 0.05 to0.10 moles/L of NaH₂PO₄ having a pH of 6.5 and preferably to 0.06moles/L of NaH₂PO₄, having a pH of 6.5.

For working it up further, this solution is subjected to an anionexchange chromatography. For this purpose, the anion exchanger isequilibrated with 0.01 to 0.05 moles/L of NaH₂PO₄ of pH 6.5 andpreferably with 0.03 moles/L of NaH₂PO₄ having a pH of 6.5, and thediafiltered protein solution is diluted with water for injectionpurposes (WFI), so that the salt concentration here also is 0.01 to 0.05moles/L of NaH₂PO₄ at a pH of 6.5 and preferably 0.03 moles/L of NaH₂PO₄at a pH of 6.5. Under these conditions, a crude immunoglobulin Gfraction passes through the column without being bound, while all otherproteins bind to the chromatographic phase.

The bound proteins are eluted with a buffer of high salt concentration(0.02 moles/L of NaH₂PO₄, 1.0 moles/L of NaCl, at a pH of 6.5).

The crude immunoglobulin G fraction optionally is filtered through avirus filter, concentrated by means of ultrafiltration or diafiltrationand re-buffered to 0.01 to 0.05 moles/L of sodium acetate of pH 5.5 andpreferably to 0.02 moles/L of sodium acetate of pH 5.5.

This adjusted crude fraction consists to the extent of more than 99% ofimmunoglobulin G. However, it is contaminated with proteolytic enzymes.

These can be removed with octanoic acid by the method of EP 0447585B1.For this purpose, the conditioned solution is treated with 0.4 to 1.5%by volume and preferably 0.8 to 1.0% by volume and especially with 0.8%by volume of octanoic acid and filtered with addition of calciumchloride. Subsequently, viruses are inactivated with 0.3% of tri-n-butylphosphate and 1% of Tween 80. At the conclusion of the reaction time,the solution is adjusted with water for injection purposes to aconductivity of 0.02 moles/L of sodium acetate, pH 5.0 buffer.

The reagents of the octanoic acid treatment and of the virusinactivation, as well as the IgG aggregates, contained in the solution,are removed by chromatography on a cationic exchanger. For this purpose,the adjusted immunoglobulin G solution is added to the equilibratedcationic exchange column and washed with 0.02 moles/L of sodium acetateat a pH of 5.0 and the immunoglobulin G fraction is eluted with abuffer, consisting of 0.02 moles/L of sodium acetate, 0.3 moles/L ofsodium chloride, and a pH of 5.0. The column is purified with 0.02moles/L of sodium acetate, 1.0 moles/L of sodium chloride, having a pHof 5.0, and 1.0 moles/L of sodium hydroxide.

The immunoglobulin G fraction is diafiltered against 0.3 moles/L ofglycine of pH 5.0 and concentrated to a protein concentration of 50 g/L.

The i.v. compatible IgG preparation of high purity can also be worked upanalogously by other known methods.

The intravenously compatible immunoglobulin G preparations, so produced,have some advantages over the conventional solutions, which usually areobtained by precipitations from plasma. For example, the IgG sub-classcomposition corresponds to the natural distribution, which occurs inplasma. The IgA content is less than 0.15% and the IgM content less than0.02% of the total protein content. The proportion of IgG aggregates isless than 0.25%. Possible impurities, which can lead to incompatibilityreactions, such as pre-kallikrein activator, pre-kallikrein, kallikrein,kininogens, plasmin, plasminogen and factor XI cannot be detected. Allother analytical parameters correspond to the European Pharmacopoeia fori.v. immunoglobulin G preparations.

Accordingly, the preparation, so obtained, fulfills all criteria anddiffers from the known product, on the one hand, by the higher IgGproportion and, on the other, with respect to the sub-class composition.

b) Albumin, Antithrombin III Transferrin

Antithrombin III (AT-III), transferrin and albumin, for example, can beobtained from the inventively produced Fraction 1.

A corresponding method is shown in FIG. 4 and comprises essentiallyaffinity chromatography, anion exchanges chromatography, virusinactivation and conventional filtering, concentrating and sterilizingsteps.

Accordingly, antithrombin III can be obtained from the startingmaterials containing it, namely the ultrafiltered and diafilteredFraction 1 by means of affinity chromatography on a heparin carrier.Such a procedure is described in the EP-A 844 254 as a preliminarycleaning step for obtaining antithrombin III, which is freed fromimpurities, a heat treatment and/or a metal chelation treatment beingcarried out subsequently there. In the inventive method, the affinitychromatography can accordingly also be carried out. A salt solution oflow concentration, such as 0.1 to 0.2 moles/L sodium chloride solution,leads to the binding of antithrombin III and a higher saltconcentration, such as 1.0 to 2.0 moles/L sodium chloride solution leadsto elution. Fraction 1 can thus be adjusted to a buffer medium of 0.02moles/L of NaH₂PO₄, 0.150 moles/L sodium chloride solution and a pH of7.0 and applied to the affinity phase.

Albumin and transferrin pass through the column without being bound. Thecolumn is washed with 0.02 moles/L of NaH₂PO₄ and 0.4 moles/L of NaCl ata pH of 7.0. Subsequently, the AT-III, bound to the heparin carrier, iseluted with 0.02 moles/L of NaH₂PO₄ and 2.0 moles/L of NaCl at a pH of7.0. The AT-III solution obtained is re-buffered to physiologicalconditions and can be processed further to a therapeutically usablepreparation by known methods, such as virus filtration andpasteurization.

The fraction, passing through the affinity chromatography column andcontaining albumin and transferrin, can be separated into transferrin bythe method of DE 3733181C1 and albumin according to the method of EP0402205B1 or EP 0792887A1. For example, after re-buffering theabove-mentioned run-through filtration of the affinity chromatography to0.02 moles/L of tris and 0.030 moles/L of sodium chloride at a pH of7.0, the solution can be applied to an anion exchanger. The transferrinis collected as a run-through fraction and virus-inactivated by knownmethods and processed further. The albumin fraction, subsequently elutedfrom the anion exchanger with 0.02 moles/L of tris and 1.0 moles/L ofsodium chloride at a pH of 7.0, can be adjusted, for example, to aconductivity of 1.8 mS/cm with a sodium acetate buffer pH of 6.0 andsubjected once again to anion exchange chromatography. Any traces oftransferrin present are removed by changing the pH to a value of 5.2.The albumin is eluted from the anion exchanger at a pH of 4.5 and aconductivity of 1.8 mS/cm. The product is processed further to atherapeutically usable albumin preparation by known methods.

Known materials are used for all the chromatographic steps employed inthe above-mentioned methods. As cation exchanger, for example,CM-Accell®, SP-Spherodex®, SP-Trisacryl-LS® or Fraktogel-TSK-SP 650®,Poros HS® or S-HyperDF® or SOURCE-305®, CM HyperDF® can be used and thesalt concentration adjusted correspondingly. As anion exchanger, forexample, QMA-Accell®, DEAE-Spherosil® or DEAE-Sepharose®, Poros HQ®,Q-HyperDF® and SOURCE-30Q® can be used. Further suitable materials areCM-Accell®, SP-Spherodex-M® or phases, produced on the basis ofsynthetic polymers, such as SP-Trisacryl-LS® and CM-Trisacryl-LS®. Forexample, heparin-Sepharose® or heparin-immobilized synthetic polymers,such as Toyopearl AF-Heparin 650M® can be used for the affinitychromatography and the above-mentioned compositions can also be selectedas buffers.

Preferably, chromatographic media based on synthetic polymers, such asthose commercially obtainable under the trade names of Poros®, SOURCE®,Macroprep®, TSK®, Toyopearl® and Hyper D®, are used in the inventivemethod. Said phases, substituted as anion exchangers, cation exchangers,hydrophobic interaction matrix and affinity matrix, are available.Tertiary or quaternary amino groups form anion exchanges, sulfoalkylgroups or carboxyalkyl groups form cation exchangers, and substituents,such as alkyl or phenyl or heparin groups, form hydrophobic phases oraffinity matrices. The appropriate buffer media and elution conditionsfor these are known to those skilled in the art.

Because of the relative resistance to pressure, high flow rates arepossible even if the particle size is small. With that, the processesdescribed can be carried out economically in a short time and with ahigh throughput. Moreover, the chromatographic phases offer theadvantage that they are chemically inert to sterilizing reagents andtherefore particularly suitable for the production of pharmaceuticalproducts.

For the sterilization, treatments with beta-propiolactone, TNBP/Tween,TNBP/NaCholate, TNBP, optionally in combination with UV radiation orvirus filtration, can be employed. The filters used, such as Planova®,Virosolv® and UltriporDD50®, are known.

The products, produced pursuant to the invention in this manner, can bestored in liquid or lyophilized form.

The advantage of the inventive method lies therein that said startingmaterials can be purified rapidly and easily so that the pure productsobtained can be obtained without significant losses in yield andcorrespond essentially to their natural composition.

The invention is explained by the following examples.

EXAMPLE 1

As starting material, 5.4 L of a human plasma, freed from clottingfactors, is used. The clotting factors are removed by known methods byseparation of the cryo-precipitate and by absorption of the PPSB factorsat DEAE-Sephadex-A50®.

Ammonium sulfate (1.2 moles/kg) is added to this pretreated plasma,which is then stirred for 5 hours at room temperature, after which waterfor injection purposes (WFI) is added until the conductivity reaches avalue of 112 mS/cm (20° C.), which corresponds to the conductivity of a0.9 molar ammonium sulfate solution, and stirring is continued for 6 to12 hours. After addition of 2% (w/w) of filter aid (such as standardSuper Cell), stirring is continued for 1 hour, after which the solutionis clarified over a depth-filter, such as a Seitz Supra 80P.

A steel column (373 mL), filled with TSK-Phenyl 5PW® is equilibratedwith 0.9 moles/L of ammonium sulfate and 0.01 moles/L of NaH₂PO₄ havinga pH of 7.0. The prepared and filtered plasma solution is applied inportions of 370 mL at a flow rate of 70 mL/min. At the end of theapplication, the column is washed with five column volumes of theequilibrating buffer and this fraction is collected (Fraction 1). Duringthe collection of Fraction 1, an ultrafiltration on a 10 KD membrane,such as Omega®, Pall-Filtron is carried out at the same time, thepermeate being recycled to the reservoir for the equilibrating buffer.

After that, Fraction 2 is obtained with an elution buffer, consisting of0.3 moles/L of ammonium sulfate and 0.01 moles/L of NaH₂PO₄ at a pH of7.0 and employed in an amount of four times the capacity of the column.The elution buffer is prepared from the equilibration buffer (0.9moles/IL of ammonium sulfate and 0.01 moles/L of NaH₂PO₄ having a pH of7.0) and a 0.01 moles/L solution of NaH₂PO₄ having a pH of 7.0, using amixing valve. For this purpose, the two buffers are mixed on-line in aratio of 1:3.

The lipoprotein fraction (Fraction 3) is dissolved from the column byadding 3 volumes of a 0.01 moles/L NaH₂PO₄ buffer, having a pH of 7.0,and discarded.

After a purification step with 3 volumes of a 1-mole/L sodium hydroxidesolution, the column is washed with WFI and equilibrated once again.

For working up all of the filtered protein solution used, 26 cycles arerequired, Fractions 1 and 2 in each case being collected in a container.After that, sterile filtration of the separated Fractions 1 and 2 iscarried out.

Yield, immunoglobulin G in Fraction 2 90.3% Yield, albumin in Fraction 1 100%

Composition of Fractions 1 and 2 in the Capillary Zone ElectrophoresisFraction 1 Fraction 2 Gamma-globulin (%) 0 61.5 β-globulin (%) 6.1 13.5α₁-globulin (%) 4.3 2.1 α₂-globulin (%) 7.7 13.3 albumin (%) 81.9 1.8fibrinogen (%) 0 7.6

EXAMPLE 2

The procedure is the same as that of Example 1.

The protein solution, freed from clotting factors, treated with ammoniumsulfate and filtered, is chromatographed, as described in Example 1, ona TSK Phenyl 5PW® column.

After Fraction 1 is eluted, the immunoglobulins and lipoproteins areeluted in one fraction from the column with 0.01 moles/L of NaH₂PO₄having a pH of 7.0. The fractions obtained show the following yields:

Fraction 1: albumin yield  100% Fraction 2: immunoglobulin G yield 95.5%

EXAMPLE 3

The procedure is the same as that of Example 1.

The protein solution, freed from clotting factors, treated with ammoniumsulfate and filtered, is chromatographed on a 3 L column (180×250 mm),filled with Toyopearl-Phenyl 650M®.

The procedure corresponds to that of Example 1. Per cycle, 2.4 L of thestarting solution are processed at a flow rate of 300 mL/min. With thiscolumn, three cycles are required for working up 4 L of plasma.

As in Example 1, three fractions are collected.

The fractions obtained show the following yields:

Fraction 1: albumin yield 100% Fraction 2: immunoglobulin G yield  96%

EXAMPLE 4

The procedure is the same as that of Example 1.

The protein solution, freed from clotting factors, treated with ammoniumsulfate and filtered, is chromatographed on a 2.5 L column, filled withToyopearl-Phenyl 650M®.

The procedure corresponds to that of Example 2.

Per cycle, 2.4 L of the starting solution are fractionated a flow rateof 125 mL/min into two fractions. For this purpose, 3 cycles arerequired.

The fractions obtained show the following yields:

Fraction 1: albumin yield 100% Fraction 2: immunoglobulin G yield  95%

EXAMPLE 5

The procedure of Examples 1, 2, 3 or 4 is followed. Instead of apolyvalent starting plasma, a pre-selected human plasma with a hightiter for the cytomegalovirus (anti-CMV) is used and processed asdescribed in Examples 1, 2, 3 or 4.

The fraction 2 obtained therefrom shows the following yields:

Yield of immunoglobulin G 90.5% Yield of anti-CMV-titer   78%

EXAMPLE 6

The procedure of Examples 1, 2, 3 or 4 is followed. Instead of apolyvalent starting plasma, a pre-selected human plasma with a hightiter for hepatitis B virus (anti-HBs) is used and processed as inExamples 1 or 2 or 3 or 4.

The fraction 2 obtained therefrom shows the following yields:

Yield of immunoglobulin G 92.0% Yield of anti-HBs-titer   82%

EXAMPLE 7

The procedure of Examples 1, 2, 3 or 4 is followed. Instead of apolyvalent starting plasma, a pre-selected human plasma with a hightiter for anti-D is used and processed as in Examples 1 or 2 or 3 or 4.

The fraction 2 obtained therefrom shows the following yields:

Yield of immunoglobulin G 91.2% Yield of anti-D titer   54%

EXAMPLE 8

An immunoglobulin G-containing Fraction 2, prepared as in Example 1, isadjusted by means of ultrafiltration and diafiltration to 0.06 moles/Lof NaH₂PO₄, a pH of 6.5 and a protein concentration of about 50 g/L.This is followed by a dilution with water for injection purposes (WFI)in the ratio of 1:2. After stirring for 1 hour, the fraction is filteredto clarify and to sterilize it.

A steel column (383 mL), filled with an anion exchanger, such as PorosHQ50®, is equilibrated with a buffer of 0.03 moles/L of NaH₂PO₄ having apH of 6.5.

The conditioned, immunoglobulin G-containing solution (500 mL) isapplied to the column at a flow rate of 120 mL/min. The column is washedwith 10 volumes of the equilibrated buffer and theimmunoglobulin-containing fraction is collected.

The remaining bound proteins are eluted from the column with 0.02moles/L of NaH₂PO₄ and 1.0 moles/L of NaCl at a pH of 6.5. For cleaningthe column, the latter is washed with 2 volumes of 1 mole/L of sodiumhydroxide and subsequently equilibrated once again.

The total amount of 2.5 L of the starting solution is chromatographed in5 cycles.

The immunoglobulin G-containing fraction, which is applied and runthrough, is filtered through a virus filter, such as Planova 35,re-buffered by means of ultrafiltration and diafiltration to 0.02moles/L of sodium acetate and a pH of 5.5 and adjusted to a proteinconcentration of about 40 g/L. To this solution, 0.8% (v/v) of octanoicacid is added and, while stirring for 1 hour, the pH is held at 5.5 with1 moles/L of sodium hydroxide. After that, 0.05 moles of calciumchloride are added, the pH is corrected to a value of 5.5 with 1 mole/Lof sodium hydroxide and stirring is continued for 2 hours.

Subsequently, the solution is treated with 2% (w/v) of filter aid, suchas standard Super Cell and filtered through a clarifying filter, such asSeitz Supra 80P. The filter cake is washed twice with 100 mL of 0.02moles/L of sodium acetate having a pH of 5.5.

The filtered solution is treated with 0.3% of TNBP (tri-n-butylphosphate) and 1% Tween 80 (Polysorbate 80) and stirred for at least 8hours at room temperature.

At the end of the reaction time, the conductivity is adjusted bydilution with WFI to that of a 0.02 moles/L sodium acetate solutionhaving a pH of 5.0 and the pH is corrected with 10% acetic acid to avalue of 5.0.

A steel column (388 mL), filled with an anion exchanger, such as PorosHS50, is equilibrated with a buffer of 0.02 moles/L sodium acetatehaving a pH of 5.0.

The conditioned immunoglobulin-G solution (2.15 L) is applied to thecolumn at a flow rate of 120 mL/min. The column is washed with 10volumes of equilibrating buffer, in order to remove the reagents of theoctanoic acid and solvent detergent treatment. This washing liquidfraction is discarded.

The bound immunoglobulin G is eluted from the column with 0.02 moles/Lof sodium acetate and 0.3 moles/L of sodium chloride at a pH of 5.0 andcollected.

The column is cleaned with 0.02 moles/L of sodium acetate and 1.0 mole/Lof sodium chloride at a pH of 5.0, followed by 1.0 mole of sodiumhydroxide.

After that, the column is equilibrated again. The total amount of 2.3 Lof the starting solution is chromatographed in 2 cycles.

By means of ultrafiltration and diafiltration against 0.3 moles/L ofglycine having a pH of 5.0, the immunoglobulin G fraction is adjusted tobe isotonic, concentrated to a protein content of 50 g/L and filtered tosterilize it.

Yield of immunoglobulin G: 74%

Analytical Data of a 5% Immunoglobulin G Solution Anti-complementaryactivity  0.68 CH₅₀/mg of protein Immunoglobulin A  0.14% ImmunoglobulinM  0.02% Sub-class: IgG₁  57.6% IgG₂  33.3% IgG₃  5.5% IgG₄  3.6%cellulose acetate electrophoresis 100% gamma-globulin pre-kallikreinactivator neg. pre-kallikrein neg. kallikrein neg. kininogen neg.plasmin neg. plasminogen neg. Factor XI neg.

The HPSE chromatogram is given in FIG. 5

EXAMPLE 9

An albumin-containing Fraction 1, prepared as in Example 1, is adjustedby means of ultrafiltration and diafiltration to 0.02 moles/L of NaH₂PO₄and 0.15 moles/L of sodium chloride having a pH of 7.0.

A 50 mL column (1.5×25 cm), filled with AF-Heparin Toyopearl®, isequilibrated with 0.02 moles/L of NaH₂PO₄ and 0.150 moles/L of sodiumchloride having a pH of 7.0.

The albumin-containing fraction (100 mL) is applied at a flow rate of 7mL/min to the column. The column is rinsed with 5 volumes of theequilibration buffer and this albumin-containing fraction is collected.

The column is washed with 0.02 moles/L of NaH₂PO₄ and 0.4 moles/L ofNaCl having a pH of 7.0 and this fraction is discarded. The AT-III iseluted with a buffer consisting of 0.02 moles/L of NaH₂PO₄ and 2.0moles/L of sodium chloride having a pH of 7.0. This fraction isre-buffered by means of ultrafiltration/diafiltration to PBS,concentrated and sterilized.

The analytical date of the AT-III solution obtained is as follows:

AT-III yield 91% protein 1.1 g/L AT-III activity 805% d.N. AT-IIIantigen 1.0 g/L albumin <0.006 g/L

The HPSE chromatogram is given in FIG. 6.

TABLE 1 Determination of the Ammonium Sulfate Concentration for theHydrophobic Interaction Chromatography Protein IgG IgA IgM Albumin (g/L)(g/L) (g/L) (g/L) (g/L) Starting point 26.7 3.59 0.78 0.34 15.8 0.5moles 26.7 3.59 0.78 0.34 15.8 ammonium sulfate 0.7 moles 26.7 3.59 0.780.34 15.8 ammonium sulfate 0.8 moles 26.7 3.59 0.78 0.34 15.8 ammoniumsulfate 1.0 mole 26.0 3.40 0.72 0.35 14.8 ammonium sulfate 1.2 moles24.9 1.93 0.38 0.21 15.7 ammonium sulfate 1.4 moles 22.0 0.73 0.16 0.1014.4 ammonium sulfate

TABLE 2 Example of an Inventive Hydrophobic Interaction Chromatographyof Human Plasma at Different Ammonium Sulfate Concentrations 0.7 Moles0.8 Moles 1.0 Moles Starting point 100 100 93.7* Albumin (%) IgG (%) 100100 94.7* Fraction 1 >95.0 >95.0 >95.0 Albumin (%) IgG (%) 34.4 <2.6<2.0 Fraction 2 1.4 1.8 3.3 Albumin (%) IgG (%) 57.2 >95.0 >95.0*Precipitation by ammonium sulfate

TABLE 3 Distribution of Relevant Plasma Protein in Fractions ofHydrophobic Interaction Chromatography Starting point (%) Fraction 1 (%)Fraction 2 (%) IgG 100 <5 >90 IgA 100 <2 >95 IgM 100 <5 >90 Albumin100 >95 <2 Transferrin 100 >95 <2 AT-III 100 >90 <10 α₁-anti-trypsin100 >90 <5 Lipids/lipoproteins 100 <5 >70Caption for FIG. 5:HPSE Chromatogram of an Immunoglobulin G Solution, Prepared According toExample 8Caption for FIG. 6:HPSE Chromatogram of an Antithrombin (AT-III) Solution, Produced byExample 8

1. A method for fractionating plasma or serum, said method comprisingsubjecting a starting solution, containing plasma or serum, to afractionation by hydrophobic interaction chromatography without rivanolprecipitation, wherein during said hydrophobic interactionchromatography a stepwise salt gradient is employed to obtain (at least)one immunoglobulin-containing fraction and one albumin-containingfraction.
 2. The method of claim 1, wherein the starting solutioncontains a plasma or serum of human origin.
 3. The method of claim 2,wherein the starting solution contains polyvalent human plasma.
 4. Themethod of claim 2, wherein the starting solution contains selected humanplasma, selected with respect to viral, bacterial or antibodies,directed against cellular antigens.
 5. The method of claim 1, wherein anammonium sulfate gradient is used for the chromatography.
 6. The methodof claim 5, wherein the chromatography comprises a first fractionationstep at a high concentration of ammonium sulfate, and a secondfractionation step at a lower concentration of ammonium sulfate.
 7. Themethod of claim 6, wherein the high concentration of ammonium sulfate isbetween 0.6 and not more than 1.4 moles/L and the lower concentration isbetween 0 and 0.4 moles/L.
 8. The method of claim 6, wherein the highconcentration of ammonium sulfate is 0.7 to 1 moles/L, and the lowerconcentration is between 0 to 0.3 moles/L.
 9. The method of claim 1,wherein the starting solution and the chromatography solid phase areadjusted to a desired high salt-gradient concentration at the start ofthe fractionation.
 10. The method of claim 1, wherein the startingsolution contains plasma, from which the clotting factors of the PPSBcomplex have been removed.
 11. The method of claim 1, wherein thestarting solution contains plasma or serum from which clotting factorVIII has been removed.
 12. The method of claim 1, wherein, afterobtaining a first fraction, two further fractions are obtained by meansof step gradients.
 13. The method of claim 12, wherein, after the firstfraction, the fractionation commences with an ammonium sulfate bufferhaving a concentration of 0.4 to 0.1 moles/L, which is then lowered toless than 0.1 to 0 moles/L.
 14. The method of claim 1, whereinphenyl-substituted or alkyl-substituted phases, based on copolymers ofglycidyl methacrylate and ethylene glycol dimethacrylate, copolymers ofpolystyrene or divinylbenzene or silica, coated with dextran orpolymers, are used as hydrophobic interaction solid phase.
 15. Themethod of claim 14, wherein copolymers of glycidyl methacrylate andethylene glycol dimethacrylate are used as hydrophobic interaction solidphase.
 16. The method of claim 14, wherein the fractionation employs ahigh concentration of ammonium sulfate buffer of 0.8 to 1.0 moles/L anda lowered concentration of ammonium sulfate of 0.3 to 0 moles/L.
 17. Themethod of claim 16, wherein a first fraction is obtained at an ammoniumsulfate concentration of 0.9 moles/L and, after that, a step gradient isemployed, the ammonium sulfate concentration initially being 0.3 moles/Land then lowered to 0 moles/L.
 18. The method of claim 1, wherein afirst fraction obtained is worked up and therapeutically usableantithrombin III, transferrin and/or albumin are obtained.
 19. Themethod of claim 18, wherein the first fraction obtained is worked up byaffinity chromatography followed by anion exchange chromatography andvirus inactivation as well as filtering, concentrating and sterilizingsteps.
 20. The method of claim 1, wherein a second faction obtained isworked up and therapeutically usable immunoglobulin is obtained.
 21. Themethod of claim 20, wherein the second fraction obtained is worked up byanion exchanger chromatography, virus inactivation, octanoic acidtreatment, as well as cation exchanger chromatography and filtering,sterilizing and concentrating steps into a compatible immunoglobulin Gpreparation.
 22. The method of claim 20, wherein the therapeuticallyusable immunoglobulin is IgG.
 23. A therapeutic method comprising thefollowing steps: a) carrying out the method of claim 1 to obtain atherapeutically usable immunoglobulin preparation, an antithrombin IIIpreparation, an albumin preparation or a transferrin preparation; and b)administering a therapeutically effective amount of at least one of saidpreparation to a patient in need thereof.